The three critical events in the pathogenesis of varicella-zoster virus (VZV) infection and its transmission are viremia, cutaneous replication and neural latency. Our overall objectives are to define regulatory/tegument gene requirements for each of these pathogenic phases using in vitro methods and our SCIDhu model of VZV skin and T cell tropism, to develop a SCIDhu neural cell model to study VZV neurotropism and to further characterize the attenuated vaccine Oka (V-Oka) virus. We will use human neural implants established within the anterior chamber of the SCID mouse eye as the primary system for VZV neurotropism studies. As an alternative, we will explore VZV inoculation of SCID animals engrafted with neuronal stem cells. Development of one or both models will make it possible to assess whether mutations altering VZV replication in skin or T cells also affect VZV neurotropism in vivo. Investigations of regulatory/tegument proteins will focus on the immediate early (IE) proteins, IE62, IE63 and IE4. ORF62 encodes the major viral transactivator of VZV and IE63 appears to have accessory transactivating activity. We have shown that one copy of IE63 is essential and have made a single copy IE63 recombinant; work is in progress to generate a single copy ORF62 recombinant. These single copy constructs will be used to introduce ORF62 and ORF63 mutations into the viral genome using parent Oka (P-Oka) cosmids. Mutagenesis targets will be selected by mapping sites of IE62tlE63 interaction and identifying putative functional regions of ORFs 62 and 63 from sequence motifs or by conservation in alphaherpesvirus genes. Domains will be defined as essential or dispensable for replication in cell culture. We propose to characterize domains in IE4 protein related to IE62 binding, dimerization, transactivation, and nuclear/cytoplasmic localization. These analyses will define IE4 regions that must be intact for infectivity. Viable recombinants that have targeted mutations in IE62, IE63 or IE4 will be evaluated for effects on VZV replication in differentiated human cells in vivo in the SCIDhu model. In order to further investigate V-Oka attenuation, chimeric recombinants made from V-Oka and P-Oka cosmids will be evaluated in the SCIDhu skin and T cell xenografts. Finally, we will exploit the model to examine structural characteristics of P-Oka virions and to compare P-Oka and V-Oka virions. Using these experimental approaches, it should be possible to create VZV recombinants that lack the capacity to disseminate by infecting T cells, or to establish persistent infection in neural cells, while retaining the capacity to replicate in skin. A better understanding of the genetic mechanisms that are required for VZV virulence in skin, T cells and neural cells will guide the design of 'second generation' live attenuated varicella vaccines. PERFORMANCESITE(S) (organization,city,state) Stanford University Medical Center, Palo Alto, California State University of NY at Buffalo, Amherst, New York University of Iowa, Iowa City, Iowa KEY PERSONNEL.Seeinstructions. Usecontinuationpagesas neededtoprovidetherequiredinformationinthe format shownbelow. Startwith PrincipalInvestigatorL.istallotherkey personneiln alphabeticaolrder,lastnamefirst. Name Organization Roleon Project Ann M. Arvin, M.D. Stanford University Medical Center P.I. John Hay, M.D. State University of NY at Buffalo Co-Investigator Charles Grose, MD. University of Iowa Co-Investigator William T. Ruyechan State University of NY at Buffalo Co-Investigator DisclosurePermissionStatement.Applicableto SBIPJSTTROnly. Seeinstructions[.] Yes [] No o PHS398 (Rev.05/01) Page_2 FormPage2 a [] Principal Investigator/Program Director (Last, first, middle): Arvin, Ann M. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page, RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,